Hepatitis B virus (HBV) is a major cause of chronic viral hepatitis that increases dramatically the risk of liver cancer and other end-stage liver diseases such as cirrhosis. HBV belongs to the Hepadnaviridae, a family of para-retroviruses that have a small DNA genome and replicate through an RNA intermediate (the pregenomic RNA, or pgRNA), by a unique reverse transcription pathway. The initiation of reverse transcription in HBV is accomplished via a novel protein priming mechanism whereby the viral reverse transcriptase (RT) serves as a specific protein primer. Following protein priming, the immature (containing pgRNA) nucleocapsid (NC) carries out reverse transcription, leading to the conversion of pgRNA to the characteristic, relaxed circular (RC) DNA. The DNA-containing (mature) NC is selectively enveloped and secreted extracellularly as virions. A host- derived protein kinase is packaged by the viral nucleocapsids and its role in viral replication remains unclear. Using recently established cell-free and cell culture systems and newly developed approaches and models, we propose to (1) define the viral and host requirements for HBV protein priming; (2) determine the role of the packaged host CDK2 in NC uncoating and infection; and (3) determine the mechanisms of regulation of HBV virion morphogenesis. Novel and specific models regarding HBV protein priming, NC uncoating, and virion formation will be tested. Recent studies suggest that effective antiviral interventions in chronic viral hepatitis ca block or reverse virus-induced liver damage and its associated liver diseases. Unfortunately, current therapies for HBV infections are very limited. The elucidation of the viral and host factor that modulate three critical stages of viral replication and assembly, i.e., the protein-primed initiation of reverse transcription, NC uncoating, and virion formation, will not only provide important new insights into mechanisms of hepadnavirus replication and virus-host interactions but also facilitate the development of novel and effective anti-HBV strategies targeted at these factors.